Chemical Constituents, Antioxidant, and Antimicrobial Activity of Allium Chinense G

Rhetso et al. Future Journal of Pharmaceutical Sciences (2020) 6:102 Future Journal of https://doi.org/10.1186/s43094-020-00100-7 Pharmaceutical Sciences

RESEARCH Open Access Chemical constituents, antioxidant, and antimicrobial activity of Allium chinense G. Don Thejanuo Rhetso, R. Shubharani, M. S. Roopa and V. Sivaram*

Abstract Background: Allium chinense G. Don is a medicinal and aromatic herb belonging to the family Amaryllidaceae, characterized by a high saponin content. The previous report has mostly been focused on the bulb, and there is very limited work on the leaf. The information about biological and chemical constituent of A. chinense leaf is still inadequate in contrast to the investigations reported on the bulb. To the best of our knowledge, there is no report on the hexane extract of both bulb and leaf extract. Therefore, the present investigation was focused on identifying and characterization of the hexane extracts of A. chinense bulb and leaf quantitatively and by using the GC-MS method and to know its scavenging, antibacterial, and antifungal activity. Results: Twenty-eight bioactive compounds were identified in the bulb and nine in the leaf extract by GC-MS analysis. The versatile compounds present in the bulb are 2-methyloctacosane (21.30%), tetracontane (14.05%), eicosane, 10-methyl (12.06%), heneicosane (8.46%), octadecyl trifluoroacetate (6.48%), and 1-heneicosanol (5.76%), whereas in the leaf, it was phytol (35.76%), tetratetracontane (18.49%), perhydrofarnesyl acetone (14.76%), and heptadecane, 2,6-dimethyl (10.79%). In quantitative estimation, saponins were estimated to have the highest with 375.000 ± 0.577 mg/g in the leaf and 163.750 ± 0.433 mg/g in the bulb. The DPPH antioxidant scavenging activity was found to be minimum in both the bulb (IC50 = 678.347 μg/ml) and leaf (IC50 = 533.337 μg/ml). A. chinense extracts of both leaf and bulb exerted potential antibacterial effects against Staphylococcus aureus and Pseudomonas aeruginosa. Leaf extract exhibited greater antifungal activity than the bulb against Aspergillus niger. Conclusion: From the analysis, the hexane leaf extract exhibited higher antibacterial, antifungal, and antioxidant activity than the bulb. Their superior activity might be due to the higher content of total saponin and terpenes. This result will lead to further in-depth research towards the potential use of this plant; the bio-constituents can be further isolated and used in medical and therapeutic applications. Keywords: Bulb extract, Leaf extract, GC-MS, Scavenging activity, Antibacterial, Antifungal

Background constituents of the plant can be used for drug develop- Plants and their bioactive compounds are a potential ment in pharmacology and medicine. source of medicine and are suitable with the prevailing The genus Allium belongs to the family Amaryllida- demands for safe and effective treatment. Traditional ceae has 500 species and are characterized by their rich knowledge procured over years of observation and inter- content of organo-sulfur as their main bioactive com- action with the environment is a substantial source of pounds [1]. Allium chinense (Fig. 1) is cultivated in the modern medicine. Isolated and purified bioactive North-Eastern part of India region; the whole plant is edible raw or cooked and is used in culinary as a flavor- * Correspondence: [email protected] ing agent. The plant has a strong onion-like aroma and Department of Botany, Bangalore University, Bengaluru, Karnataka 560056, pungent taste, the entire plant is an expectorant, India

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Rhetso et al. Future Journal of Pharmaceutical Sciences (2020) 6:102 Page 2 of 9

aqueous methanol extract [7] and laxogenin extracted from hot water extract [8] from A. chinense bulb have anti-tumor property. The protective effects of steroids from 60% ethanol bulb extract of A. chinense have great potential to prevent cardiac injuries induced by oxidative stress [9]. On literature survey, most of the earlier works have been carried out on the bulb and the biological and chemical report on A. chinense leaf is still scarce. How- ever, no report to date has been found on hexane leaf and bulb extract of A. chinense. Therefore, the objective of the study was to characterize the chemical profile of the bulb and leaf hexane extract using GC-MS and the second objective was to determine the scavenging activity and antimicrobial activity of A. chinense.

Methods Collection of plant material Fresh and healthy plants of A. chinense were collected from the farmers in Kohima district, Nagaland, India. The collected plants were washed, carefully shade-dried till all the moisture contents were removed and ground to a fine powder using an electric grinder and stored in an airtight glass bottle at room temperature 27 ± 2 °C.

Extraction A. chinense leaf and bulb were extracted separately using a soxhlet extractor with 200 ml hexane for 18–20 h. The extracts were separated from the solvent using a rotary evaporator at 30–40 °C and stored at 4 °C for further use.

Quantitative analysis Estimation of total alkaloid content The total alkaloids were determined using the method described by Tan [10]. Atropine was used as a standard for plotting the calibration curve. The extracts (1 mg/ml) Fig. 1 A. chinense were dissolved in 2 N HCl and washed with chloroform, the layers were separated using a separating funnel, later bromocresol green solution and phosphate buffer (pH carminative, and astringent also used to treat bronchitis, 4.7) were added and the mixture was vortexed, the yel- diarrhea, and angina pleurisy [2]. The bulb has high me- low color complex formed at the bottom was pipetted dicinal value in traditional Chinese medicine for over out to measure the absorbance at 470 nm. The total al- thousands of years and is the major source of the “Xie- kaloid content was estimated using the linear regression bai” drug used in treating thoracic pain, stenocardia equation obtained from the standard graph of atropine heart, asthma, and stagnant blood [3]. Among the many further mean ± SD (n = 3) was calculated and expressed bioactive compounds present in A. chinense, the import- as mg/g atropine equivalent (AE). ant active compounds are steroidal saponins, amino acids, sulfur, nitrogen, and flavonoids compound [4]. Estimation of total flavonoids The earlier study on A. chinense leaf essential oil re- Estimation of total flavonoids content was done using ported 34 organo-sulfur-containing compounds account- the aluminum trichloride (AlCl3) method with different ing 94% of the total volatiles [5]. The chemical concentrations of quercetin as standard [11]. Extracts of investigation carried out in essential oil of the bulb re- 1 mg/ml were diluted with distilled water and 5% ported a profuse content of sulfide compounds, as high NaNO2 solution was added. After being incubated for 6 as 97.71% [6]. Spirostanol saponins extracted from 60% min, 0.15 ml of AlCl3 10% solution was added followed Rhetso et al. Future Journal of Pharmaceutical Sciences (2020) 6:102 Page 3 of 9

by 2 ml of 10% NaOH, the final volume was adjusted to Table 1 Quantitative analysis of A. chinense bulb and leaf 5 ml using distilled water, and it was then incubated at extracts room temperature for 15 min. Absorbance was read at Sl. Quantitative Linear regression Leaf Bulb 510 nm. All analysis was carried out in triplicate times No. analysis (mg/g) equation for standard and the total flavonoid content was calculated using lin- 1 Alkaloids Y = 0.0003X + 0.0009, R2 77.000 ± 77.830 ± ear regression equation obtained from the standard plot = 0.9996 (atropine) 0.333 0.289 2 of quercetin and mean ± SD (n = 3) was calculated. The 2 Flavonoids Y = 0.0029X + 0.0031, R 6.862 ± 9.275 ± = 0.9992 (quercetin) 0.597 0.398 result was expressed as mg/g quercetin equivalent (QE). 3 Phenol Y = 0.0127X + 0.0252, R2 18.409 ± 20.100 ± = 0.9991 (gallic acid) 0.273 0.069 Estimation of total phenolic content 4 Saponins Y = 0.0002x + 0.003, R2 = 375.000 163.750 The total phenolics were estimated with Folin- 0.999 (quillaia) ± 0.577 ± 0.433 Ciocalteu’s method [12]. Gallic acid at varying concen- Values are mean ± SE (n = 3) trations was used as standard; plant extracts (1 mg/ml), 5 ml of FC (1:10) diluted reagent, and 4 ml sodium bicar- The calibration curve for inhibition was prepared and bonate (7.5%) were taken in a test tube and the mixture IC50 values were calculated. was shaken and incubated in the dark for 30 min at 20 °C. The absorbance was read at 765 nm, all the ana- Antimicrobial analysis by disk diffusion method lysis was performed thrice. The total phenolic content The antimicrobial activity of the bulb and leaf extract was estimated using the linear regression equation from was screened by the agar disk diffusion method. Muller- the standard calibration curve further mean ± SD (n = Hinton agar (MHA) (Hi-Media) plates were swabbed 3) was calculated and expressed as mg/g gallic acid with gram-positive Staphylococcus aureus (MTCC916) equivalent (GAE). and gram-negative Pseudomonas aeruginosa (MTCC741) bacteria and fungi Aspergillus niger (MTCC281) in Estimation of saponins Sabouraud dextrose agar (SDA) plate [15]. Plant extracts The saponin content of the extracts was measured as de- of 1 mg/ml concentration were prepared in 0.1% di- scribed by Le et al. [13]. Quillaia was used to obtain the methyl sulfoxide (DMSO). Four wells measuring 6 mm standard calibration curve. The sample (1 mg/ml) was were bored in the inoculated media with a sterile cork- mixed with 500 μL of 8% vanillin and 500 μL of 72% sul- borer. The disks were loaded with 30 μl of the extract furic acid and incubated at 60 °C for 10 min after cooling and placed on the MHA and SDA plate. The Petri-plates to room temperature absorbance was recorded at 544 were further incubated at 37 °C for 24 h for bacteria and nm. The total saponin content was quantified using the 22 °C for 48 h for fungi. At the end of incubation, zones linear regression equation obtained from the standard of inhibition (ZOI) were observed around the disk and graph of quillaia further mean ± SD (n = 3) was quanti- measured with a transparent ruler in millimeters. fied as mg/g quillaia equivalent (QE). All determination Streptomycin (30 μg/ml) and fluconazole (1 mg/ml) were was carried out in triplicates. used as the positive control for bacteria and fungi re- spectively. 0.1% DMSO was used as a negative control. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay GC-MS analysis The ability of A. chinense bulb and leaf extracts to scav- To obtain the complete chemical profile, analysis was enge free radicals was assessed according to the standard performed using GC-MS (SHIMADZU QP2010S) sys- DPPH method [14]. Ascorbic acid was used as the tem, with the Rxi-5Sil MS column; length 30 meters, standard and the assay was performed in triplicate. One and helium (99.99%) as a carrier gas at a flow of 1.00 milliliter of the varying concentrations (1–100 μg/ml) was added to 3.0 ml of freshly prepared DPPH (0.06 Table 2 Antioxidant activity measured by DPPH scavenging mM). The mixture was further incubated in the dark for Concentration (μg/ml) Ascorbic acid Leaf extract Bulb extract 15 min at room temperature (27 ± 2 °C). The decrease in 1 9.630 ± 0.057 1.238 ± 0.133 1.972 ± 0.010 the absorbance was measured at 517 nm using a UV-Vis 10 15.313 ± 0.067 3.665 ± 0.007 2.369 ± 0.056 spectrophotometer. The percentage of inhibition was 25 29.390 ± 0.115 5.077 ± 0.015 4.317 ± 0.012 calculated using the following formula: 50 49.127 ± 0.037 7.502 ± 0.018 6.594 ± 0.032 75 62.160 ± 0.034 9.036 ± 0.139 7.479 ± 0.010 DPPH radical scavenging activity ðÞ¼% ½ÂðÞA0 − A1 =A0 100 100 83.167 ± 0.072 10.729 ± 0.260 8.731 ± 0.006 where A0 is absorbance of control (DPPH) and A1 ab- IC50 55.118 533.337 678.347 sorbance of extracts Values are mean % inhibition ± SE (n = 3; p < 0.05) Rhetso et al. Future Journal of Pharmaceutical Sciences (2020) 6:102 Page 4 of 9

Fig. 2 Scavenging activity of ascorbic acid, bulb, and leaf extract of A. chinense mL/min. Injection mode was splitless and sampling time Results 2.00 min; flow control mode was linear velocity at a split Quantitative analysis ratio 100.0 MS and ID 0.25 mm. The ion source Phytochemicals have been analyzed quantitatively. temperature was set at 200.00 °C, interface temperature The total alkaloid, flavonoid, phenol, and saponin 280.00 °C, and oven temperature was programmed at concentration of the bulb and leaf extract are pre- 70.0 °C. Solvent cut time was 6.50 min, start time 7.00 sented in Table 1. min, end time 35.75 min, event time 0.50 s, and scan range 50–500 m/z. DPPH assay The activity of A. chinense extract against free radicals is shown in Table 2 and Fig. 2.TheA. chinense Identification of compounds extract showed less scavenging capacity both in the The identification of chemicals was based on the peaks leaf (IC50 = 533.337 μg/ml) and bulb (IC50 = of the compounds at different mass-to-charge ratios. Re- 678.347 μg/ml) when compared to standard ascorbic sults were obtained in accordance with the mass spectral acid (IC50 = 55.118 μg/ml) with significant difference library of National Institute of Standards and Technol- (p < 0.05). ogy, NIST-11. The unknown spectrum was compared with the standard spectrum existing in the database of Antimicrobial activity the WILEY 8 library. Antimicrobial activity was observed in all the species tested (Table 3,Fig.3). The extracts of A. chinense exhibited exquisite antibacterial activity with 15 nm Statistical analysis (leaf) and10 nm (bulb) ZOI in gram-positive All the estimation was carried out in triplicates. The data (Staphylococcus aureus) and 20 mm (leaf) and 16 mm obtained in this study were analyzed using a one-way (bulb) ZOI in gram-negative bacteria (Pseudomonas analysis of variance (ANOVA) by using Prism V. 5.00 aeruginosa). The absence of ZOI was interpreted as (Graphpad Inc. USA). The results were expressed as the absence of activity. In Aspergillus niger,bulbex- mean ± SE. Significance value (P) < 0.05 was selected as tract showed 7 mm ZOI and 14 mm ZOI in the leaf a point of minimum significance rate. extract.

Table 3 Diameter of zones of inhibition (mm) of bulb and leaf extract of A. chinense against tested microorganisms Sample/test organism Staphylococcus aureus (gram-positive) (mm) Pseudomonas aeruginosa Aspergillus niger (gram-negative) (mm) (mm) Leaf 15 20 14 Bulb 10 16 7 Positive control 16 18 16 Negative control NA NA NA NA no activity Rhetso et al. Future Journal of Pharmaceutical Sciences (2020) 6:102 Page 5 of 9

Fig. 3 Growth inhibition of S. aureus, P. aeruginosa, and A. niger caused by A. chinense bulb (Hex-1) and leaf (Hex-2) extracts

GC-MS analysis cyclopropane, 1,1-dichloro-2,2,3,3-tetramethyl-, 1- henei- GC-MS analysis is a superior technique to identify the cosanol, 1-heptacosanol, 2-methyloctacosane, octadecyl phytoconstituents. Nine compounds were identified from trifluoroacetate, eicosane, 7-hexyl, n-tetracosanol-1, tetra- hexane leaf extract and twenty-eight compounds from tetracontane, docosane, 11-butyl-, heneicosane, n- hexane bulb representing 100% of the total extract. The nonadecanol-1, and vitamin E. identification was based on retention time, peak area, molecular formula, molecular weight, and molecular struc- Discussion ture which are shown in (Figs. 4 and 5, Tables 4 and 5). In the quantitative estimation of A. chinense bulb and The GC-MS analysis of hexane leaf extract revealed the leaf, high content of saponin was observed. Saponin pos- presence of this compounds viz., perhydrofarnesyl acet- sesses antibiotic, insecticidal, and fungicidal activity [16]. one, phytol, nonadecane, 2-methyl-, heptadecyl heptaflur- Steroidal saponins isolated from A. chinense bulb possess obutyrate, eicosane, 7-hexyl-, octadecyl trifluoroacetate, anti-tumor property [7]. Saponins are cytotoxic and in- gamma-tocopherol, tetratetracontane, heptadecane, 2,6- hibit the migration ability of B16 and 4T1 cells [4]. Lax- dimethyl- and the compounds identified in the bulb are 9, ogenin a saponin isolated from A. chinense bulb have 12 octadecadienoic acid, methyl ester, tritetracontane, 1- anti-tumor property in stage-two lung carcinogenesis ethylsulfanylmethyl-2,8,9-trioxa-5-aza-1-sila-bicyclo[3.3.3] [8]. Therefore, A. chinense can be used as a potential undecane, 3,6,9,12-tetraoxahexadecan-1-ol, 9,12-octadeca- plant for the extraction of saponin for medicinal and dienoic acid, ethyl ester, 3,4,4a,5,6,7,8,8a-octahydro-spiro[- pharmaceutical use. cyclohexane-1,4’-(2H-1,3-benzothiazine)]-2-thione-8a-ol, Alkaloids have been reported to have anticancer, heptadecane, 4,8,12,16-tetramethylheptadecan-4-olide, antibacterial, antiviral, and antifungal activity [17]. hexadecanoic acid, 2-methylpropyl ester, nonadecane, 2- Aclidinium bromide a drug from alkaloid is used to methyl-, hexadecanoic acid,3-[(trimethylsilyl)oxy] propyl treat chronic obstructive pulmonary disease; atropine ester, hexadecanoic acid-2-hydroxyl-1- (hydroxy methyl) and scopolamine are alkaloid derivatives used in ethyl ester, eicosane, 10-methyl-, diisooctyl phthalate, n- traditional medicine for treating asthma [18]. A. chi- propyl 9,12-octadecadienoate, eicosane, 2-methyl-, nense showed a moderate content of alkaloids which

Fig. 4 GC-MS chromatogram of hexane extract of A. chinense leaf Rhetso et al. Future Journal of Pharmaceutical Sciences (2020) 6:102 Page 6 of 9

Fig. 5 GC-MS chromatogram of hexane extract of A. chinense bulb

could be exploited for their pharmacological [19, 24, 27]. The leaf extract has an outstanding properties. antibacterial activity against gram-negative bacteria Low content of phenols [19] and flavonoids [20]in- P. aeruginosa. dicate low antioxidant activity. Phenols are known to The result of the current study supports the use of have anti-inflammatory, antimicrobial, anesthetic, anti- both bulb and leaf hexane extracts as a potential oxidant, anti-tubercular, anticancer, analgesic, and antibacterial and antifungal; the leaf extract pre- anti-Parkinson activity [21]. Flavonoids are known to sented as a wide antimicrobial spectrum compared have antioxidant effects and have been shown to in- to the bulb. hibit the initiation and progression promotion of tu- The GC-MS characterization revealed that the com- mors [22]; consumption of flavonoids decreases pounds found in both the bulb and leaves extract coronary heart disease [23]. were not reported earlier in this plant. The difference DPPH is a stable free radical with absorption spec- in results reported in essential oil [6]maybedueto tra at 517 nm and loses its ideal absorption accepting the extraction method, soil pH, seasonal variation, cli- an electron, resulting in a change from purple to yel- matic conditions, and many other factors. low color, displaying the scavenging potential [24]. In the leaf extract, phytol was the major component The low or negligible antioxidant capacity in the hex- identified with a concentration of 35.76% and reten- ane leaf and bulb extract may be due to the low con- tion time of 20.475 min which is an acyclic diterpene tent of phenol [19] and flavonoid [20]. Our findings with anticancer, anti-diuretic, nematicide, hepatopro- correlate with the previous report by Lin in A. chi- tective, hypocholesterolemic, anticoronary, anti- nense bulb [25]. androgenic antimicrobial, antioxidant, antiarthritic, The leaf extract showed higher antibacterial activ- anti-inflammatory, antidiabetic, and immunostimula- ity (20 mm) than the standard (18 mm) against P. tory [27] followed by tetratetracontane with a peak aeruginosa, it is known that gram-negative bacteria area of 18.49% appeared at 33.630 min is a long chain arehighlyresistanttomanyantibiotics. The absence alkane, having antibacterial activity [28]. Perhydrofar- of ZOI was interpreted as the absence of activity. nesyl acetone with 14.76% was the first compound The activities are expressed as resistant if ZOI was detected in the leaf at 17.148 min and is a sesquiter- less than 7 mm, intermediate (8–10 mm), and sensi- penoid that has extensive biological activities such as, tive if more than 11 mm [26]. On observing the zone antimicrobial, allelopathic, cytotoxic, and antifeedant of inhibition, the bulb exhibited resistant activity activity [29]. whereas leaf was highly sensitive as an antibacterial. The GC-MS analysis of the bulb displayed alkane as In A. niger, bulb extract showed intermediate (7 mm) the major group. In the 28 compounds revealed, the activity whereas sensitive activity with 14 mm ZOI dominance of compounds such as 2-methyloctacosane was observed in the leaf extract. The high compos- with the peak area of 21.30% appeared at 27.601 min ition of terpene, viz. phytol (35.76%) and perhydro- which is an alkane with antimicrobial [30]. Tetratetra- farnesyl (14.7%) in the leaf and 2-methyl octacosane contane with a peak area of 14.05% which appeared (21.30%) in the bulb exhibited antimicrobial activity at 30.497 min is an alkane having antibacterial Rhetso et al. Future Journal of Pharmaceutical Sciences (2020) 6:102 Page 7 of 9

Table 4 Phytocompounds identified in the hexane extract of A. chinense leaf by GC-MS analysis

property [28]. Eicosane, 10-methyl with a peak area of containing compounds were identified in the crude 12.06% appeared at 24.850 min, an alkane which has extract. high antioxidant [31]. Heneicosane with a peak area of 8.46% reported at 33.939 min is an aliphatic hydro- Conclusion carbon; it belongs to the higher alkane group having The quantitative analysis showed a high content of sapo- bio pesticidal property [32]. nins in the leaf extract. GC-MS analysis of the extract The GC-MS revealed that the bulb and leaves have suggests that numerous medicinally important bioactive a different bio-constituent; only three compounds tet- constituents are present in A. chinense leaf and bulb, ratetracontane, nonadecane, and 2-methyl-octadecyl with high terpene content in the leaf and alkanes in the trifluoroacetate appeared in both the extract. Our bulb. findings on the chemical composition of hexane bulb These important findings revealed through our and leaf extract did not correlate with the previous study suggest isolation of bioactive compounds, reports on the essential oil [5, 6]andnosulfide- and screening its activity will have tremendous Rhetso et al. Future Journal of Pharmaceutical Sciences (2020) 6:102 Page 8 of 9

Table 5 Phytocompounds identified in the hexane extract of A. benefits, considering its availability, that it is edible chinense bulb by GC-MS analysis and has medicinal value, and that it is non-toxic; however, a toxicological analysis would be of ne- cessity to develop safe drugs. This work represents an initial step to understand the plant phytochemical constitution which could facilitate further investigation.

Abbreviations AlCl3: Aluminum trichloride; AE: Atropine equivalent; DMSO: Dimethyl sulfoxide; DPPH: 2,2-Diphenyl-1-picrylhydrazyl; FC: Folin-ciocalteu; GAE: Gallic acid equivalent; GC-MS: Gas chromatography-mass spectroscopy; HCl: Hydrochloric acid; MHA: Mueller-Hinton agar; NA: No activity; NaOH: Sodium hydroxide; NaNO2: Sodium Nitrite; NIST: National Institute of Standards and Technology; QE: Quercetin equivalent; QE: Quillaia equivalent; SDA: Sabouraud dextrose agar; SE: Standard error; UV-Vis: Ultraviolet-visible; ZOI: Zone of inhibition

Acknowledgements The authors are thankful to the Department of Botany, Bangalore University, Bangalore 560056, for providing facilities, and Central Instrumentation Unit, Kerala Forest Research Institute (KFRI), Peechi, Thrissur, Kerala, India, for GC- MS analysis.

Plant authentication The plant was authenticated by Dr. V Rama Rao, Regional Ayurveda Research Institute for Metabolic Disorders, Central Council for Research in Ayurvedic Sciences, Ministry of AYUSH, Govt. of India, Bengaluru 560109. The authenticated sample was preserved with the specimen voucher number RRCBI-mus 244.

Authors’ contributions TR performed all the above analysis and drafted the manuscript. SR participated in performing the analysis and interpretation of the data. RMS participated in performing the analysis. VS participated in the design, drafting, and critically revising the manuscript. All authors have read and approved the manuscript.

Funding The first author is thankful to the University Grant Commission (UGC) Maulana Azad National Fellowship (MANF) of the Government of India for providing Senior Research Fellowship (SRF).

Availability of data and materials The data used to analyze the findings of this study are available from the corresponding author upon request.

Ethics approval and consent to participate Not applicable.

Consent for publication Not applicable.

Competing interests The authors declare that they have no competing interests.

Received: 9 March 2020 Accepted: 31 August 2020

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